Automated selection of microorganisms and identification using maldi

ABSTRACT

A method and apparatus for locating and selecting a colony of microorganisms on a culture dish and identifying microorganisms in said selected colony using MALDI. The method comprises the automated steps of locating and selecting a colony of microorganisms on a culture dish; obtaining a sample of said selected colony of microorganisms; depositing at least some of said sample of said selected colony of microorganisms on a target plate; and transferring said target plate with said sample in an apparatus for performing MALDI for identification of said sample of said selected colony of microorganisms. A sample of a colony of microorganisms is automatically deposited on a depositing spot such that the sample covers at most approximately half of said one of the depositing spots of the target plate. A suspension of a sample of microorganisms is automatically prepared by automatically picking the sample with a picking tool and submerging the picking tool with said sample in a suspension, after which the picking tool is vibrated in vertical sense only to release the sample from the picking tool.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/388,430, filed on Sep. 26, 2014, which is a national phaseentry under 35 U.S.C. §371 of International Application No.PCT/NL2013/050239 filed Apr. 2, 2013, published in English, which claimspriority from U.S. application Ser. No. 61/618,003 filed Mar. 30, 2012,all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention generally relates to a method for locating andselecting a colony of microorganisms and identifying microorganismsusing MALDI, in particular MALDI-TOF-MS (Matrix Assisted LaserDesorption and Ionization Time-of-Flight Mass Spectroscopy) and thesystems for performing such methods.

MALDI-analysis is a useful tool for solving structural problems inbiochemistry, immunology, genetics and biology. Samples are ionized inthe gas phase and a time of flight (TOF) analyzer is used to measure ionmasses. TOF analysis begins when ions are formed and are accelerated toa constant kinetic energy as they enter a drift region. They arrive at adetector following flight times that are proportional to the square rootof their masses. A mass spectrum is created because ions of differentmass arrive at the detector at different times.

Mass spectrometry can be a powerful tool in the fields of drug discoveryand development, genotyping, and proteome research. In addition MALDIhas already been used for characterization and identification ofbacteria and microorganisms. Current trends in research are to analyzelarger and larger numbers of samples using quantities of individualsamples ranging from the micro-mole levels to ato-mole levels. As aresult, samples are also becoming smaller and needs exists for efficientand reliable acquisition of the correct amount of micro-organisms andaccurately depositing a sample of the acquired amount on a target plateused in the MALDI-instrument.

In a typical MALDI TOF MS operation, the sample to be analyzed isspotted or deposited on a metal plate (also called target plate orMALDI-plate), reagents are added (matrix) that support ionization, andthen they are dried to form crystals. In these instruments, the targetplate is positioned in a fixed position in the MALDI-instrument. Thetarget plate has a plurality of depositing spots (e.g. from 24 to 384depositing spots on a single target plate) and these depositing spotshave a fixed orientation with regard to the edges of the target plate.The target plate is positioned on an X-Y stage so that an obtainedsample of a colony of microorganisms can be deposited on a selecteddepositing spot. A high voltage potential is maintained between thetarget plate and a metal grid. This voltage can be maintained or pulsed,depending upon the desired results and a vacuum is created in thechamber. A laser is fired into the sample/matrix and a plume of ions isformed. The voltage difference is used to accelerate the ions up aflight tube so that they can be analyzed. The analysis directly relatesthe time of flight to the mass of the ionized component.

Several parameters can affect the quality of the results, includingflatness of the target, amount and type of matrix, concentration of thesample, conductivity of the sample target, accuracy of placement on thedepositing spot, as well as other variables.

In particular, an important aspect is the handling of the sample and theconcentration of the sample. It is known that a suspension is made froma sample of a colony of microorganisms and that a researcher pipettes adrop of the obtained suspension containing the sample onto a depositingspot of the target plate by hand. To provide a correct analysis,however, the suspension has to contain a sufficient concentration of thesample from the start.

In preparing such a suspension of a sample of microorganisms, a handhelddevice comprising a drive. The drive comprises a housing with a rotarydrive motor contained therein, and a connector which is configured forthe releasable attachment of the sample acquisition device to the drive.The sample acquisition device comprises a sample collection region whichis first brought into contact with biological material (mostly grown ona culture dish) to be analyzed. Thereafter the sample collection regionis attached to the rotary drive, brought into contact with a liquidmedium contained in a tube and the rotary drive is activated for acertain period of time such that a sample of the biological material inthe sample acquisition device is released in the liquid medium. Afterthe sample collection region is removed from the tube the suspensiontube holds a suspension containing the sample which for example can beused for performing MALDI-analysis.

However, the efficiency of release of the sample from the sampleacquisition region can in some cases be insufficient to perform acorrect analysis of the microorganism suspended in the liquid medium.This may result in a prepared suspension that is unusable, resulting inlost of time and money. In addition since the sample collection regionis rotated within the tube the tube must be of a sufficient dimension toallow such rotation without the sample acquisition region contacting theinner wall of the tube which might be detrimental for the release of themicroorganism in the liquid medium. Such a relative large dimensionedtube thus contains a relatively large volume of liquid medium, leadingto a corresponding lengthy time of further processing such liquidsuspension medium. For example, the time in which the liquid suspensioncontaining the sample has to incubate in an incubator is proportional tothe amount of suspension liquid. Therefore, there is a need for a methodin which the preparation of a suspension of a sample of microorganismsis performed automatically leading to a much more reproducible manner ofpreparing such a suspension. In addition there is a need for reliablyand reproducibly releasing the sample on a depositing spot of a targetplate.

BRIEF SUMMARY OF THE INVENTION

In order to solve at least one of the problems mentioned above thepresent invention provides a method for locating and selecting a colonyof microorganisms on a culture dish and identifying microorganisms insaid selected colony using MALDI, wherein said method comprises theautomated steps of: locating and selecting a colony of microorganisms ona culture dish; obtaining a sample of said selected colony ofmicroorganisms; depositing at least some of said sample of said selectedcolony of microorganisms on a target plate; and transferring said targetplate with said sample in an apparatus for performing MALDI foridentification of said sample of said selected colony of microorganisms.It appears that the problems mentioned above for the greater part findtheir cause therein that the steps are performed manually and thesesteps are therefore prone to undesired variance and mistakes, leading toincorrect results from the MALDI-instrument, additional costs and lossof time. By automating each of the steps these problems can be overcometo at least a large extent. In the present field it has been taken forgranted that at least some of the steps only could be performedmanually, however in contrast hereto the present invention provides thepossibility for the first time to automate all the steps necessary forlocating and selecting a colony of microorganisms and identifyingmicroorganisms in said selected colony using MALDI.

In an embodiment of a method according to the invention the step ofdepositing at least some of said sample of said selected colony ofmicroorganisms on a target plate is performed by directly placing saidobtained sample of said selected colony of microorganisms on said targetplate. By directly placing a sample on a target plate there is no needto first prepare a suspension of said sample, thereby circumventingevery problem which might arise from preparing such a suspension.Preferably the method comprises the further automated step of overlayinga drop of a MALDI matrix solution on the portion of the sample depositedon the target plate.

In an alternative embodiment of a method according to the invention thestep of depositing at least some of said sample of said selected colonyof microorganisms on a target plate is performed by the automated stepsof: transferring said obtained sample into a suspension tube containingan amount of a suspension medium; preparing a sample suspension bytransferring at least a portion of said obtained sample into saidsuspension medium; obtaining a drop of said sample suspension; andtransferring said drop of sample suspension onto said target plate. Bycompletely automating the preparation of a suspension the inventionprovides an accurate and reproducible method of using suspensions foridentifying microorganisms using MALDI. In particular when the methodfurther comprises the automated step of overlaying a drop of a MALDImatrix solution on said drop of sample suspension deposited on thetarget plate, the inventive method is extremely suitable forcharacterization of microorganisms. The best analysis results can beobtained in a further embodiment of a method according to the inventionin which said drop of sample suspension deposited on the target plate isallowed to dry before said drop of MALDI matrix solution is overlaid.This alternative method of using a suspension is furthermore extremelyuseful in case another test or analysis is to be performed on the sampleof the colony of microorganisms. Such additional analysis can in aparticularly reproducible and efficient manner be realized in anembodiment of a method according to the invention in which the methodfurther comprises the automated steps of: obtaining a second drop ofsaid sample suspension; depositing said second drop of sample suspensionon a test culture dish; and transferring said test culture dish to anapparatus for performing a susceptibility test or another additionalanalysis. Consequently the inventive method can be used to automaticallyobtain or pick a sample which can be fed into available ID/ASTinstruments including but not limited to BACTEC™, Phoenix, MGIT,BacT/Alert.

The present invention also provides the possibility of performing suchadditional analysis in case no suspension is made from an obtainedsample. In this situation a particular advantageous embodiment of amethod according to the invention further comprises the automated stepsof: obtaining a second sample of a colony of microorganisms from theculture dish; transferring said second sample of said selected colony ofmicroorganisms; depositing at least some of said second sample of saidselected colony of microorganisms on a test culture dish; andtransferring said test culture dish to an apparatus for performing asusceptibility test or another additional test.

Since for performing characterization and identification ofmicroorganisms normally a plurality of colonies is grown on a culturedish, it is furthermore important that a sample is obtained from acolony of interest. If samples are taken from non-interesting coloniesthe efficient use of time and the MALDI-instrument is compromised.According to the knowledge of the applicant up till now no automatedprocess or apparatus exists for discriminating interesting colonies fromnon-interesting ones. However, the process of discriminating coloniescan be at least partly automated under maintaining a very high certaintyof correct discrimination in an embodiment of a method in accordancewith the invention in which the method comprises before the automatedstep of locating and selecting a colony of microorganisms on a culturedish, the step of providing a culture dish comprising a number ofcolonies of microorganisms, obtaining an initial image of said culturedish including all the colonies of microorganisms, displaying saidinitial image of said culture dish including all the colonies ofmicroorganisms on a display, and selecting at least one colony ofmicroorganisms in said initial image. In this manner a researcher oranalyst can select colonies of interest based on thorough education andknowledge. In a particular embodiment said culture dish is provided withan individual identification identifying said culture dish, such as abar code, and the method further comprises the step of storing saidinitial image of said culture dish including all colonies, storinginformation regarding said at least one selected colony ofmicroorganisms, storing said identification of said culture dish in amemory of a central control computer. In an additional embodiment theresearcher or analyst can manually enter processing instructionsregarding the processing to which a selected colony of microorganisms ofsaid culture dish is to be subjected, said processing instructions beingstored in said memory of said central control computer for later use.

In a further embodiment of the invention the method comprises theautomated steps of positioning said culture dish on a stage for aculture dish, obtaining an image of said culture dish positioned in saidstage, obtaining the identification of said culture dish, comparing theimage obtained by the imaging device of said picking tool device withthe stored initial image of said culture dish for obtaining informationregarding the location of the selected colony of microorganisms andoptionally for obtaining the processing instructions regarding theprocesses to be performed on said selected colony of microorganisms. Bycomparing the image of the culture dish when it is placed in the pickingtool device with the initial image, the location of the selectedcolonies can be obtained automatically, for example by computerizedimage comparison.

In still a further embodiment of a method according to the invention themethod comprises the step of automatic preparing a suspension of asample of microorganisms comprising the steps of:

-   -   providing a first picking tool and providing a positioning        device with a picking tool holder for holding a picking tool,        said positioning device being arranged for positioning a picking        tool in a starting position above the obtained location of the        selected colony of microorganisms on the culture dish and for        automatically lowering and raising a picking tool towards and        away from the culture dish and for positioning a picking tool in        a transfer position, respectively;    -   positioning said first picking tool in said picking tool holder        of the positioning device;    -   by said positioning device positioning the first picking tool in        the starting position above the obtained location of the        selected colony of microorganisms on the culture dish,        automatically lowering the first picking tool towards the        culture dish into contact with the microorganism to pick up a        sample of said microorganism, automatically raising the first        picking tool with said sample of the microorganism away from the        culture dish to the transfer position;    -   providing a suspension tube holder for holding a suspension        tube;    -   positioning a suspension tube in the suspension tube holder;    -   providing an automatic suspension medium dispenser for        automatically dispensing a suspension medium in a suspension        tube held in the suspension tube holder;    -   by the automatic dispenser automatically supplying an initial        amount of suspension medium into the suspension tube held in the        suspension tube holder;    -   providing a transferring device for automatically transferring a        picking tool from the transfer position of the positioning        device to a position above a suspension tube held in the        suspension tube holder, and for lowering and raising a picking        tool into and away from a suspension medium contained in a        suspension tube, and for positioning a picking tool in a waiting        position above a suspension tube held in the suspension tube        holder, respectively;    -   by said transferring device automatically transferring said        first picking tool with the sample of the microorganism from the        transfer position of the positioning device to a position above        the suspension tube held in the suspension tube holder, lowering        said first picking tool with the sample of the microorganism        into the suspension medium contained in the suspension tube; by        the transferring device oscillating the first picking tool in a        linear vertical movement for a period of time while the first        picking tool with the sample of said microorganism is submerged        in the suspension medium; and after said period of time has        elapsed raising the first picking tool away from the suspension        medium contained in the suspension tube to the waiting position;    -   providing a turbidity meter for performing measurements of the        turbidity of a suspension medium contained in a suspension tube        held in the suspension tube holder;    -   at least after the period of time during which the picking tool        is oscillated has elapsed measuring the turbidity of the        suspension medium contained in the suspension tube held in the        suspension tube holder by the turbidity meter and providing a        final measurement value indicative of the measured turbidity;    -   providing a controller communicatively connected to the        positioning device, the transferring device, the automatic        suspension medium dispenser and the turbidity meter for        automatically controlling the movement of the positioning        device, the movement of the transferring device, the operation        of the automatic suspension medium dispenser and the operation        of the turbidity meter, respectively;    -   by said controller:        -   a) determining whether the final measurement value is above            a first threshold value previously stored in a memory of the            controller, if yes perform step b); or whether the final            measurement value is identical to or below the first            threshold value and identical to or above a second threshold            value previously stored in the memory of the controller,            said first threshold value being equal to or greater than            the second threshold value, if yes perform step c); or            whether the final measurement value is below the second            threshold value, if yes then perform step d);        -   b) controlling the automatic suspension medium dispenser to            supply an additional amount of suspension medium into the            suspension tube;        -   c) providing a signal that the suspension tube with the            suspension can be removed from the suspension tube holder            for further processing; or        -   d) providing a further picking tool; positioning said            further picking tool in said picking tool holder of the            positioning device; by said positioning device positioning            the further picking tool in the starting position above the            culture dish, automatically lowering the further picking            tool towards the culture dish into contact with the            microorganism to pick up an additional sample of said            microorganism, automatically raising the further picking            tool with said sample of the microorganism away from the            culture dish to the transfer position; by said transferring            device automatically transferring said further picking tool            with the additional sample of the microorganism from the            transfer position of the positioning device to a position            above the suspension tube held in the suspension tube            holder, lowering said further picking tool with the            additional sample of the microorganism into the suspension            medium contained in the suspension tube; by the transferring            device oscillating the further picking tool in a linear            vertical movement for a period of time while the further            picking tool with the additional sample of said            microorganism is submerged in the suspension medium; and            after said period of time has elapsed raising the further            picking tool away from the suspension medium contained in            the suspension tube to the waiting position; at least after            the period of time during which the further picking tool is            oscillated has elapsed measuring the turbidity of the            suspension medium contained in the suspension tube held in            the suspension tube holder by the turbidity meter and            providing an additional final measurement value indicative            of the measured turbidity; and performing step a). In this            manner it is possible to prepare a suspension of a sample of            a microorganism in an advanced automatic manner while by            means of the controller and the turbidity meter it is            possible to provide a suspension tube containing a            suspension medium which contains an amount of microorganism            which is always sufficient (and reproducible) to perform a            correct analysis of the microorganism.

In an embodiment of a method for automatic preparing a suspension of asample of microorganisms according to the invention the controller isarranged such that the step of measuring the turbidity of the suspensionmedium contained in the suspension tube held in the suspension tubeholder by the turbidity meter is additionally performed during theperiod of time during which the picking tool is oscillated, wherein theturbidity meter is arranged for providing an on-line measurement valueindicative of the measured turbidity during the period of time duringwhich the picking tool is oscillated to the controller. In this way anextremely fast automatic determination of the amount of microorganism inthe suspension can be obtained. In particular, if during oscillation theon-line measurement value of the turbidity is equal to or lower than thefirst threshold value and equal to or greater than the second thresholdvalue the controller controls the movement of the transferring devicesuch that the picking tool is raised to the waiting position, and thecontroller further provides a signal that the suspension tube with thesuspension can be removed from the suspension tube holder for furtherprocessing. In this manner the oscillation of the picking tool isstopped when the suspension medium contains a sufficient amount ofmicroorganisms, so that the method can be performed extremely timeefficient.

The mutual arrangement of the pick tool and the sensors of turbiditymeter is such that during the oscillation of the pick tool the pick tooldoes not obstruct the pathway of the turbidity meter.

In a further embodiment of a method for automatic preparing a suspensionof a sample of microorganisms according to the invention the controlleris arranged such as to control the turbidity meter that the step ofmeasuring the turbidity of the suspension medium contained in thesuspension tube held in the suspension tube holder by the turbiditymeter is started before the picking tool is submerged in the suspensionmedium contained in the suspension tube. In this way it is for examplepossible to check whether the initial suspension medium used is notcontaminated. Furthermore, this provides an indication of the startingvalue for the turbidity which is useful in determining the finalmeasurement value.

In a still further embodiment of a method for automatic preparing asuspension of a sample of microorganisms according to the invention themethod further comprises the step of providing as suspension tube holderfor holding a suspension tube a rotatable suspension tube holder forrotating a suspension tube held in the rotatable suspension tube holder,the step of arranging the controller such that it is communicativelyconnected to the rotatable suspension tube holder for controlling therotation of the suspension tube holder, and the step of arranging thecontroller such that the suspension tube is rotated during measurementof the turbidity of the suspension medium contained in the suspensiontube. Such a rotation of the suspension tube enables that the turbiditymeasurements can be performed at a number of positions within thesuspension tube which are rotationally spaced from each other leading toa more correct final measurement of the turbidity of the suspension. Therotation as such is not necessary for releasing the sample from thepicking tool, the oscillational linear vertical movement of the pickingtool is more than sufficient to release the sample.

Although a further picking tool can be used that is different from thefirst picking tool, the method can be performed in an economical waywhen in step d) the first picking tool is provided as further pickingtool; and the positioning of said further picking tool in said pickingtool holder of the positioning device is performed by the transferringdevice under control of the controller.

In a still further embodiment of a method for automatic preparing asuspension of a sample of microorganisms according to the invention theadditional amount of suspension medium is determined by the controllerbased on the initial amount of suspension medium, the final measurementvalue and the value of the first and/or second threshold value. Thismakes it possible to use just enough additional suspension medium asnecessary and thus in this embodiment as less as possible suspensionmedium can be used.

Since according to the inventive method for automatic preparing asuspension of a sample of microorganisms the picking tool is oscillatedin a vertical linear movement relative to the suspension tube, thedimension of the suspension tube can be relatively small. This makes itpossible that in an embodiment of a method according to the inventionthe controller can be arranged for controlling the automatic suspensionmedium dispenser such that the supplied initial amount is about 0.5-2ml, preferably about 1 ml. Such a relatively small amount of suspensionmedium is sufficient for preparing a correct suspension of a sample ofmicroorganisms. In such a method for automatic preparing a suspension ofa sample of microorganisms it is this possible to use as suspension tubea suspension tube having a substantially circular cross-section having adiameter of about 6 to about 12 mm, preferably about 10 mm, which isrelatively small when compared to the traditional tubes which have adiameter of about 16 mm. With such a relatively small suspension tube acorrect release of the sample from the picking tool is obtained when thecontroller is arranged for controlling the oscillation of thetransferring device such that the picking tool oscillates at a frequencybetween about 5 Hz to about 120 Hz, preferably about 30 Hz to about 90Hz, most preferably about 50 Hz. Preferably the controller is arrangedfor controlling the oscillation of the transferring device such that thepicking tool oscillates with an amplitude of about 0.5 mm to about 4 mm,preferably about 2 mm to about 3 mm, which results in an optimal releaseof the sample from the picking tool. In case the controller is arrangedfor controlling the oscillation of the transferring device such that theperiod of time during which the picking tool oscillates is about 3seconds to about 10 seconds, preferably about 6 seconds, it appears thatin practically all cases the complete sample can be released from thepicking tool.

The values for the frequency, amplitude and duration are dependent onthe properties of the specific microorganism and, for example itsadherence to the picking tool. From inspection by imaging it can bededuced whether or not the sample has at least for the greater part beenreleased from the picking tool by first using the above mentionedpreferred values. If there is still some material left on the pickingtool than the vertical oscillation is repeated within the given rangesat different values.

An automated method for preparing a suspension of a sample ofmicroorganisms according to the invention can be obtained when themethod additionally comprises the step of providing an automatic culturedish positioning and removing device for automatically positioning andremoving a culture dish comprising said microorganism on and from thestage, respectively. The controller is then arranged such that it iscommunicatively connected to the automatic culture dish positioning andremoving device for controlling the operation of the automatic culturedish positioning and removing device. In this manner positioning aculture dish comprising said microorganism on the stage can be performedautomatically under control of the controller. A further automation isrealized by providing an automatic suspension tube positioning andremoving device for automatically positioning and removing a suspensiontube in and from the suspension tube holder, respectively. Thecontroller is then arranged such that it is communicatively connected tothe automatic suspension tube positioning and removing device forcontrolling the operation of the automatic suspension tube positioningand removing device, so that positioning of a suspension tube in thesuspension tube holder can be performed automatically under control ofthe controller. Advantageously the controller is then arranged such thata culture dish is allowed to be automatically removed from the stage bythe automatic culture dish positioning and removing device only afterthe signal that the suspension tube with the suspension can be removedfrom the suspension tube holder for further processing has beenprovided. Furthermore it is then preferred that the controller isarranged such that a suspension tube holder is automatically removedfrom the suspension tube holder by the automatic suspension tubepositioning and removing device only after the signal that thesuspension tube with the suspension can be removed from the suspensiontube holder for further processing has been provided.

In still a further embodiment of a method according to the invention,the method comprises the step of providing an identifying mark on thesuspension tube, and the step of storing the identifying mark of saidsuspension tube together with the properties of the suspension with alink to the identity of the culture dish from which the selected colonyof microorganisms was obtained in the memory of said central controlcomputer the method can not only be automatically operated in anextremely efficient manner but also the correct and fast processing ofthe obtained analysis results is improved.

In a still further embodiment of a method according to the invention,wherein the processing instructions for a selected colony ofmicroorganisms indicate to directly place said obtained sample of saidselected colony of microorganisms on said target plate, the methodcomprises the following automated steps: providing a first picking tooland providing a positioning device with a picking tool holder forholding a picking tool, said positioning device being arranged forpositioning a picking tool in a starting position above the obtainedlocation of the selected colony of microorganisms on the culture dishand for automatically lowering and raising a picking tool towards andaway from the culture dish and for positioning a picking tool in atransfer position, respectively; positioning said first picking tool insaid picking tool holder of the positioning device; by said positioningdevice positioning the first picking tool in the starting position abovethe obtained location of the selected colony of microorganisms on theculture dish, automatically lowering the first picking tool towards theculture dish into contact with the microorganism to pick up a sample ofsaid microorganism, automatically raising the first picking tool withsaid sample of the microorganism away from the culture dish to thetransfer position; providing a target plate holder for holding a targetplate, said target plate having a plurality of depositing spots;positioning a target plate in the target plate holder; providing atransferring device for automatically transferring the picking tool fromthe transfer position of the positioning device to a position above oneof the depositing spots of the target plate, and for lowering thepicking tool such that the sample of the colony of microorganismspresent on the picking tool comes into contact with the target plate,moving the picking tool in a plane parallel to the plane of the targetplate such that the sample of the colony of microorganisms is depositedon the depositing spot, in particular covering at most approximatelyhalf of said one of the depositing spots of the target plate; andraising said picking tool from the target plate. By performing thedeposition of the sample automatically a more reproducible and accuratedeposition of the sample on the depositing spot can be obtained then bymanually depositing a sample. Furthermore it appears then when thesample covers at most approximately half of said one of the depositingspots of the target plate, the analysis results obtained from theMALDI-instrument of the part of the depositing spot which initially wasnot covered with the sample are surprisingly extremely more accuratethan the analysis results obtained from the MALDI-instrument of the partof the depositing spot which initially was covered with the sample.While the inventors do not wish to be bound by a particular theory, itis thought that the crystallization which takes place after a drop ofmatrix material has been overlaid on the sample covering part of thedepositing spot ensures that also the part of the depositing spot whichwas not covered contains an amount of sample material, and that thisamount is extremely suitable for providing excellent analysis results.In an alternative embodiment of a method according to the invention,wherein in case the processing instructions for a selected colony ofmicroorganisms indicate to obtain a drop from said sample suspension andto transfer said drop onto said target plate, the method comprises thefollowing automated steps: providing a pipetting tool in said pickingtool device and providing a positioning device with a pipetting toolholder for holding said pipetting tool, said positioning device beingarranged for positioning said pipetting tool in a starting positionabove the suspension tube and for automatically lowering and raising thepipetting tool into and out of the suspension and for positioning thepipetting tool in a transfer position, respectively; positioning saidpipetting tool in said pipetting tool holder of the positioning device;by said positioning device positioning the pipetting tool in thestarting position above the suspension tube, lowering the pipetting toolinto the suspension in said suspension tube, operating the pipettingtool to pick up an amount of suspension, raising the pipetting tool withsaid amount of suspension to the transfer position; said pipetting toolcomprising a pressurizable chamber closed by a controlled valve forcontaining the amount of suspension medium; providing a target plateholder for holding a target plate, said target plate having a pluralityof depositing spots; positioning a target plate in the target plateholder; providing a transferring device for automatically transferringthe pipetting tool from the transfer position of the positioning deviceto a position above one of the depositing spots of the target plate, andfor lowering the pipetting tool to a predefined distance above thetarget plate, pressurizing the chamber to a pressure in a range of about0.5 bar to 1.1 bar, and opening the valve for such a time that a drop ofsuspension with a volume in a range of about 0.5 to 3.0 μl is depositedon the depositing spot, in particular covering at most approximatelyhalf of said one of the depositing spots of the target plate; andraising said pipetting tool from the target plate. In dependence of theproperties of the specific microorganism, e.g. its stickiness, thepressure value and the opening time can be adjusted to obtain a smalldrop of suspension which can be reproducibly prepared and which as aresult of the automated process can be deposited accurately on thetarget plate.

In order to avoid cross-contamination in a preferred embodiment of amethod according to the invention the shape of the pipetting tool, inparticular the dispensing tip thereof, is such that depositing the dropof suspension on the target plate takes place in a splash free manner.It has appeared that depending on the kind of microorganism used, andespecially the stickiness thereof, in addition to choosing a correctpressure in the range mentioned above and opening time of the valve inthe range mentioned above a suitable shape of the pipetting tool ensuresthat a drop of suspension can be is deposited in a splash free manner.

In a further embodiment of a method according to the invention in whichthe method comprises the step of providing an identifying mark on thetarget plate and optionally providing an identifying mark on thedepositing spots of said target plate, and the step of storing theidentifying mark of said target plate and depositing spots together withthe properties of the suspension with a link to the identity of theculture dish from which the selected colony of microorganisms wasobtained in the memory of said central control computer the method cannot only be automatically operated in an extremely efficient manner butalso the correct and fast processing of the obtained analysis results isimproved.

The present invention further relates to a method for automaticpreparing a suspension of a sample of microorganisms, said methodcomprises the steps of:

-   -   providing a stage for a culture dish comprising said        microorganism;    -   positioning a culture dish comprising said microorganism on the        stage;    -   providing a first picking tool and providing a positioning        device with a picking tool holder for holding a picking tool,        said positioning device being arranged for positioning a picking        tool in a starting position above the culture dish and for        automatically lowering and raising a picking tool towards and        away from the culture dish and for positioning a picking tool in        a transfer position, respectively;    -   positioning said first picking tool in said picking tool holder        of the positioning device;    -   by said positioning device positioning the first picking tool in        the starting position above the culture dish, automatically        lowering the first picking tool towards the culture dish into        contact with the microorganism to pick up a sample of said        microorganism, automatically raising the first picking tool with        said sample of the microorganism away from the culture dish to        the transfer position;    -   providing a suspension tube holder for holding a suspension        tube;    -   positioning a suspension tube in the suspension tube holder;    -   providing an automatic suspension medium dispenser for        automatically dispensing a suspension medium in a suspension        tube held in the suspension tube holder;    -   by the automatic dispenser automatically supplying an initial        amount of suspension medium into the suspension tube held in the        suspension tube holder;    -   providing a transferring device for automatically transferring a        picking tool from the transfer position of the positioning        device to a position above a suspension tube held in the        suspension tube holder, and for lowering and raising a picking        tool into and away from a suspension medium contained in a        suspension tube, and for positioning a picking tool in a waiting        position above a suspension tube held in the suspension tube        holder, respectively;    -   by said transferring device automatically transferring said        first picking tool with the sample of the microorganism from the        transfer position of the positioning device to a position above        the suspension tube held in the suspension tube holder, lowering        said first picking tool with the sample of the microorganism        into the suspension medium contained in the suspension tube; by        the transferring device oscillating the first picking tool in a        linear vertical movement for a period of time while the first        picking tool with the sample of said microorganism is submerged        in the suspension medium; and after said period of time has        elapsed raising the first picking tool away from the suspension        medium contained in the suspension tube to the waiting position;    -   providing a turbidity meter for performing measurements of the        turbidity of a suspension medium contained in a suspension tube        held in the suspension tube holder;    -   at least after the period of time during which the picking tool        is oscillated has elapsed measuring the turbidity of the        suspension medium contained in the suspension tube held in the        suspension tube holder by the turbidity meter and providing a        final measurement value indicative of the measured turbidity;    -   providing a controller communicatively connected to the        positioning device, the transferring device, the automatic        suspension medium dispenser and the turbidity meter for        automatically controlling the movement of the positioning        device, the movement of the transferring device, the operation        of the automatic suspension medium dispenser and the operation        of the turbidity meter, respectively;    -   by said controller:        -   a) determining whether the final measurement value is above            a first threshold value previously stored in a memory of the            controller, if yes perform step b); or whether the final            measurement value is identical to or below the first            threshold value and identical to or above a second threshold            value previously stored in the memory of the controller,            said first threshold value being equal to or greater than            the second threshold value, if yes perform step c); or            whether the final measurement value is below the second            threshold value, if yes then perform step d);        -   b) controlling the automatic suspension medium dispenser to            supply an additional amount of suspension medium into the            suspension tube;        -   c) providing a signal that the suspension tube with the            suspension can be removed from the suspension tube holder            for further processing; or        -   d) providing a further picking tool; positioning said            further picking tool in said picking tool holder of the            positioning device; by said positioning device positioning            the further picking tool in the starting position above the            culture dish, automatically lowering the further picking            tool towards the culture dish into contact with the            microorganism to pick up an additional sample of said            microorganism, automatically raising the further picking            tool with said sample of the microorganism away from the            culture dish to the transfer position; by said transferring            device automatically transferring said further picking tool            with the additional sample of the microorganism from the            transfer position of the positioning device to a position            above the suspension tube held in the suspension tube            holder, lowering said further picking tool with the            additional sample of the microorganism into the suspension            medium contained in the suspension tube; by the transferring            device oscillating the further picking tool in a linear            vertical movement for a period of time while the further            picking tool with the additional sample of said            microorganism is submerged in the suspension medium; and            after said period of time has elapsed raising the further            picking tool away from the suspension medium contained in            the suspension tube to the waiting position; at least after            the period of time during which the further picking tool is            oscillated has elapsed measuring the turbidity of the            suspension medium contained in the suspension tube held in            the suspension tube holder by the turbidity meter and            providing an additional final measurement value indicative            of the measured turbidity; and performing step a). In this            manner it is possible to prepare a suspension of a sample of            a microorganism in an advanced automatic manner while by            means of the controller and the turbidity meter it is            possible to provide a suspension tube containing a            suspension medium which contains an amount of microorganism            which is always sufficient (and reproducible) to perform a            correct analysis of the microorganism.

In an embodiment of a method for automatic preparing a suspension of asample of microorganisms according to the invention the controller isarranged such that the step of measuring the turbidity of the suspensionmedium contained in the suspension tube held in the suspension tubeholder by the turbidity meter is additionally performed during theperiod of time during which the picking tool is oscillated, wherein theturbidity meter is arranged for providing an on-line measurement valueindicative of the measured turbidity during the period of time duringwhich the picking tool is oscillated to the controller. In this way anextremely fast automatic determination of the amount of microorganism inthe suspension can be obtained. In particular, if during oscillation theon-line measurement value of the turbidity is equal to or lower than thefirst threshold value and equal to or greater than the second thresholdvalue the controller controls the movement of the transferring devicesuch that the picking tool is raised to the waiting position, and thecontroller further provides a signal that the suspension tube with thesuspension can be removed from the suspension tube holder for furtherprocessing.

In a further embodiment of a method for automatic preparing a suspensionof a sample of microorganisms according to the invention the controlleris arranged such as to control the turbidity meter that the step ofmeasuring the turbidity of the suspension medium contained in thesuspension tube held in the suspension tube holder by the turbiditymeter is started before the picking tool is submerged in the suspensionmedium contained in the suspension tube. In this way it is for examplepossible to check whether the initial suspension medium used is notcontaminated. This can for example be done by performing a turbiditymeasurement of a suspension tube and compare the obtained value with apredetermined value indicating that the suspension is uncontaminated. Incase the difference exceeds a threshold value, then e.g. a warningsignal can be given indicating that the suspension is contaminated.

In a still further embodiment of a method for automatic preparing asuspension of a sample of microorganisms according to the invention themethod further comprises the step of providing as suspension tube holderfor holding a suspension tube a rotatable suspension tube holder forrotating a suspension tube held in the rotatable suspension tube holder,the step of arranging the controller such that it is communicativelyconnected to the rotatable suspension tube holder for controlling therotation of the suspension tube holder, and the step of arranging thecontroller such that the suspension tube is rotated during measurementof the turbidity of the suspension medium contained in the suspensiontube. Such a rotation of the suspension tube enables that the turbiditymeasurements can be performed at a number of positions within thesuspension tube which are rotationally spaced from each other leading toa more correct final measurement of the turbidity of the suspension. Therotation as such is not necessary for releasing the sample from thepicking tool, the oscillational linear vertical movement of the pickingtool is more than sufficient to release the sample.

Although a further picking tool can be used that is different from thefirst picking tool, the method can be performed in an economical waywhen in step d) the first picking tool is provided as further pickingtool; and the positioning of said further picking tool in said pickingtool holder of the positioning device is performed by the transferringdevice under control of the controller.

In a still further embodiment of a method for automatic preparing asuspension of a sample of microorganisms according to the invention theadditional amount of suspension medium is determined by the controllerbased on the initial amount of suspension medium, the final measurementvalue and the value of the first and/or second threshold value. Thismakes it possible to use just enough additional suspension medium asnecessary and thus in this embodiment as less as possible suspensionmedium can be used. In case the measured value indicates that the amountof microorganisms is too low, then the control system controls thepicking tool to obtain an additional sample from the same selectedcolony, and the process is repeated.

Since according to the inventive method for automatic preparing asuspension of a sample of microorganisms the picking tool is oscillatedin a vertical linear movement only, the dimension of the suspension tubecan be relatively small. This makes it possible that in an embodiment ofa method according to the invention the controller can be arranged forcontrolling the automatic suspension medium dispenser such that thesupplied initial amount is about 0.5-2 ml, preferably about 1 ml. Such arelatively small amount of suspension medium is sufficient for preparinga correct suspension of a sample of microorganisms. In such a method forautomatic preparing a suspension of a sample of microorganisms it isthis possible to use as suspension tube a suspension tube having asubstantially circular cross-section having a diameter of about 6 toabout 12 mm, preferably about 10 mm, which is relatively small whencompared to the traditional tubes which have a diameter of about 16 mm.With such a relatively small suspension tubes a correct release of thesample from the picking tool is obtained when the controller is arrangedfor controlling the oscillation of the transferring device such that thepicking tool oscillates at a frequency between about 5 Hz to about 120Hz, preferably about 30 Hz to about 90 Hz, most preferably about 50 Hz.Preferably the controller is arranged for controlling the oscillation ofthe transferring device such that the picking tool oscillates with anamplitude of about 0.5 mm to about 4 mm, preferably about 2 mm to about3 mm, which results in an optimal release of the sample from the pickingtool. In case the controller is arranged for controlling the oscillationof the transferring device such that the period of time during which thepicking tool oscillates is about 3 seconds to about 10 seconds,preferably about 6 seconds, it appears that in practically all cases thecomplete sample can be released from the picking tool.

The invention further relates to an apparatus for automatic preparationof a suspension of a sample of microorganisms for performing a methodfor locating and selecting a colony of microorganisms on a culture dishand identifying microorganisms in said selected colony using MALDI orfor performing a step of a method for automatic preparing a suspensionof a sample of microorganisms according to the inventive methods, saidapparatus comprising:

-   -   a stage for a culture dish comprising said microorganism;    -   a first and a further picking tool and a positioning device with        a picking tool holder for holding a picking tool, said        positioning device being arranged for positioning a picking tool        in a starting position above the culture dish and for        automatically lowering and raising a picking tool towards and        away from the culture dish and for positioning a picking tool in        a transfer position, respectively;    -   a suspension tube holder for holding a suspension tube;    -   an automatic suspension medium dispenser for automatically        dispensing a suspension medium in a suspension tube held in the        suspension tube holder;    -   a transferring device for automatically transferring a picking        tool from the transfer position of the positioning device to a        position above a suspension tube held in the suspension tube        holder, and for lowering and raising a picking tool into and        away from a suspension medium contained in a suspension tube,        and for positioning a picking tool in a waiting position above a        suspension tube held in the suspension tube holder,        respectively, said transferring device further being arranged        for oscillating a picking tool in a linear vertical movement for        a period of time;    -   a turbidity meter for performing measurements of the turbidity        of a suspension medium contained in a suspension tube held in        the suspension tube holder and for providing a final measurement        value indicative of the measured turbidity;    -   a controller communicatively connected to the positioning        device, the transferring device, the automatic suspension medium        dispenser and the turbidity meter for automatically controlling        the movement of the positioning device, the movement of the        transferring device, the operation of the automatic suspension        medium dispenser and the operation of the turbidity meter,        respectively;    -   said controller being arranged for:        -   a) determining whether the final measurement value is above            a first threshold value previously stored in a memory of the            controller, if yes said controller being arranged for            performing step b); or whether the final measurement value            is identical to or below the first threshold value and            identical to or above a second threshold value previously            stored in the memory of the controller, said first threshold            value being equal to or greater than the second threshold            value, if yes said controller being arranged for performing            step c); or whether the final measurement value is below the            second threshold value, if yes said controller being            arranged for performing step d);        -   b) controlling the automatic suspension medium dispenser to            supply an additional amount of suspension medium into the            suspension tube;        -   c) providing a signal that the suspension tube with the            suspension can be removed from the suspension tube holder            for further processing; or        -   d) positioning said further picking tool in said picking            tool holder of the positioning device; by said positioning            device positioning the further picking tool in the starting            position above the culture dish, automatically lowering the            further picking tool towards the culture dish into contact            with the microorganism to pick up an additional sample of            said microorganism, automatically raising the further            picking tool with said sample of the microorganism away from            the culture dish to the transfer position; by said            transferring device automatically transferring said further            picking tool with the additional sample of the microorganism            from the transfer position of the positioning device to a            position above the suspension tube held in the suspension            tube holder, lowering said further picking tool with the            additional sample of the microorganism into the suspension            medium contained in the suspension tube; by the transferring            device oscillating the further picking tool in a linear            vertical movement for a period of time while the further            picking tool with the additional sample of said            microorganism is submerged in the suspension medium; and            after said period of time has elapsed raising the further            picking tool away from the suspension medium contained in            the suspension tube to the waiting position; at least after            the period of time during which the further picking tool is            oscillated has elapsed measuring the turbidity of the            suspension medium contained in the suspension tube held in            the suspension tube holder by the turbidity meter and            providing an additional final measurement value indicative            of the measured turbidity; and performing step a).

In a further embodiment of an apparatus according to the invention thecontroller is arranged for controlling the turbidity meter such thatmeasuring the turbidity of the suspension medium contained in thesuspension tube held in the suspension tube holder by the turbiditymeter is started before the picking tool is submerged in the suspensionmedium contained in the suspension tube.

In a still further embodiment of an apparatus according to the inventionthe suspension tube holder for holding a suspension tube is a rotatablesuspension tube holder for rotating a suspension tube held in therotatable suspension tube holder, wherein the controller is arranged forbeing communicatively connected to the rotatable suspension tube holderfor controlling the rotation of the suspension tube holder, and saidcontroller is arranged for rotating the suspension tube duringmeasurement of the turbidity of the suspension medium contained in thesuspension tube.

In an advantageous embodiment of an apparatus according to the inventionin step d) the first picking tool is provided as further picking tool;and said controller is arranged for controlling the transferring devicefor positioning said further picking tool in said picking tool holder ofthe positioning device.

Preferably the controller is arranged for determining the additionalamount of suspension medium based on the initial amount of suspensionmedium, the final measurement value and the value of the first and/orsecond threshold value. In particular the controller is arranged forcontrolling the automatic suspension medium dispenser such that thesupplied initial amount is about 0.5-2 ml, preferably about 1 ml. In afurther embodiment of an apparatus according to the invention thecontroller is arranged for controlling the oscillation of thetransferring device such that the picking tool oscillates at a frequencybetween about 5 Hz to about 120 Hz, preferably about 30 Hz to about 90Hz, most preferably about 50 Hz. Furthermore the controller can bearranged for controlling the oscillation of the transferring device suchthat the picking tool oscillates with an amplitude of about 0.5 mm toabout 4 mm, preferably about 2 mm to about 3 mm, and for controlling theoscillation of the transferring device such that the period of timeduring which the picking tool oscillates is about 3 seconds to about 10seconds, preferably about 6 seconds.

In an inventive device for the automatic preparation of a suspension ofa sample of microorganisms the suspension tube can have a substantiallycircular cross-section with a diameter of about 6 to about 12 mm,preferably about 10 mm.

A fully automatic device according to the invention when the apparatuscomprises an automatic culture dish positioning and removing device forautomatically positioning and removing a culture dish comprising saidmicroorganism on and from the stage, respectively, said controller beingarranged for being communicatively connected to the automatic culturedish positioning and removing device for controlling the operation ofthe automatic culture dish positioning and removing device, and forautomatically positioning a culture dish comprising said microorganismon the stage, and when the apparatus comprises an automatic suspensiontube positioning and removing device for automatically positioning andremoving a suspension tube in and from the suspension tube holder,respectively, said controller being arranged for being communicativelyconnected to the automatic suspension tube positioning and removingdevice for controlling the operation of the automatic suspension tubepositioning and removing device, and for automatically positioning asuspension tube in the suspension tube holder. In this case it is thenpreferred that the controller is arranged for allowing a culture dish tobe automatically removed from the stage by the automatic culture dishpositioning and removing device only after the signal that thesuspension tube with the suspension can be removed from the suspensiontube holder for further processing has been provided. In addition thecontroller is then preferably arranged for automatically removing asuspension tube holder from the suspension tube holder by the automaticsuspension tube positioning and removing device only after the signalthat the suspension tube with the suspension can be removed from thesuspension tube holder for further processing has been provided.

The invention still further relates to a method for automaticallydepositing a sample of a colony of microorganisms on a depositing spotof a target plate for MALDI, wherein the method comprises the step ofdepositing the sample of the colony of microorganisms on the depositingspot such that the sample covers at most approximately half of said oneof the depositing spots of the target plate. It has been discovered thatthen when the sample covers at most approximately half of said one ofthe depositing spots of the target plate, the analysis results obtainedfrom the MALDI-instrument of the part of the depositing spot whichinitially was not covered with the sample are surprisingly extremelymore accurate than the analysis results obtained from theMALDI-instrument of the part of the depositing spot which initially wascovered with the sample. It is assumed that the crystallization whichtakes place after a drop of matrix material has been overlaid on thesample covering part of the depositing spot ensures that also the partof the depositing spot which was not covered contains an amount ofsample material, and that this amount is extremely suitable forproviding excellent analysis results. The physical or chemical processeswhich are the cause for this effect are, at the moment, unclear, butperhaps more clarity might arise when the fundamental processesunderlying MALDI are known. In an embodiment of such a method the sampleof a colony of microorganisms is in the form of a suspension, whereinthe sample of the suspension is deposited on a depositing spot of thetarget plate in the form of a drop having a volume in a range of about0.5 to 3.0 μl.

The invention still further relates to a method for automaticallydepositing a drop of a suspension containing a sample of a colony ofmicroorganisms on a depositing spot of a target plate for MALDI, whereinthe method comprises the following automated steps:

-   -   providing a pipetting tool in a picking tool device and        providing a positioning device with a pipetting tool holder for        holding said pipetting tool, said positioning device being        arranged for positioning said pipetting tool in a starting        position above a suspension tube holding said suspension        containing a sample of a colony of microorganisms, and for        automatically lowering and raising the pipetting tool into and        out of the suspension and for positioning the pipetting tool in        a transfer position, respectively;    -   positioning said pipetting tool in said pipetting tool holder of        the positioning device;    -   by said positioning device positioning the pipetting tool in the        starting position above the suspension tube, lowering the        pipetting tool into the suspension in said suspension tube,        operating the pipetting tool to pick up an amount of suspension,        raising the pipetting tool with said amount of suspension to the        transfer position; said pipetting tool comprising a        pressurizable chamber closed by a controlled valve for        containing the amount of suspension medium;    -   providing a target plate holder for holding the target plate,        said target plate having a plurality of depositing spots;    -   positioning the target plate in the target plate holder;    -   providing a transferring device for automatically transferring        the pipetting tool from the transfer position of the positioning        device to a position above one of the depositing spots of the        target plate, and for lowering the picking tool to a predefined        distance above the target plate, pressurizing the chamber to a        pressure in a range of about 0.5 bar to 1.1 bar, and opening the        valve for such a time that a drop of suspension with a volume in        a range of about 0.5 to 3.0 μl is deposited on said one of the        depositing spots; and    -   raising said pipetting tool from the target plate. Preferably        the shape of the pipetting tool is such that depositing the drop        of suspension on the target plate takes place in a splash free        manner. In particular the method comprises pressurizing the        chamber to such a pressure in a range of about 0.5 bar to 1.1        bar, and opening the valve for such a time that a drop of        suspension with a volume in a range of about 0.5 to 3.0 μl is        deposited on at most half said one of the depositing spots. It        has been discovered that then when the sample covers at most        approximately half of said one of the depositing spots of the        target plate, the analysis results obtained from the        MALDI-instrument of the part of the depositing spot which        initially was not covered with the sample are surprisingly        extremely more accurate than the analysis results obtained from        the MALDI-instrument of the part of the depositing spot which        initially was covered with the sample. It is assumed that the        crystallization which takes place after a drop of matrix        material has been overlaid on the sample covering part of the        depositing spot ensures that also the part of the depositing        spot which was not covered contains an amount of sample        material, and that this amount is extremely suitable for        providing excellent analysis results. The physical or chemical        processes which are the cause for this effect are, at the        moment, unclear, but perhaps more clarity might arise when the        fundamental processes underlying MALDI are known.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further explained with reference to FIG. 1, inwhich a non-limiting exemplary embodiment of an apparatus of theinvention is shown, which FIG. 1 will also be used to explain exemplaryembodiments of the method according to the invention.

DETAILED DESCRIPTION

In the present invention MALDI or MALDI-TOF-MS is used to identifymicroorganisms. In a MALDI TOF MS operation a sample of a colony ofmicroorganisms is spotted or deposited on a target plate which is heldin a fixed position in the MALDI-instrument. Such a target plate has aplurality of depositing spots (e.g. from 24 to 384 depositing spots on asingle target plate) and these depositing spots have a fixed orientationwith regard to the edges of the target plate. The target plate ispositioned on an X-Y stage so that an obtained sample of a colony ofmicroorganisms can be deposited on a selected depositing spot, whereinthe location where a specific sample has been deposited is indicated bythe X-Y parameters and is stored in a memory of a central controlcomputer.

Although not depicted in detail in FIG. 1, the target plate ispositioned below a transfer track 18 at a position indicated by B. Asample can be transferred along the transfer track 18 from a culturedish 3 and/or a suspension tube 11 to above the target plate at positionB, where the sample is lowered to be deposited on a depositing spot ofthe target plate.

Although the invention will be described detailed below with referenceto preparing a suspension containing a sample and depositing a drop ofsaid suspension on a depositing spot of a target plate, the presentinvention also relates to directly depositing a sample obtained (picked)from a culture dish onto a depositing spot of a target plate.

In general, in the latter method a colony of microorganisms isautomatically located and detected on a culture dish. A sample of saidselected colony of microorganisms is obtained in an automated way, e.g.by a picking tool which is brought into contact with the colony. Atleast some of said sample of said selected colony of microorganisms isdirectly deposited on a target plate in an automated way, by loweringthe picking tool such that the sample of the colony of microorganismspresent on the picking tool comes into contact with the target plate,and moving the picking tool in a plane parallel to the plane of thetarget plate such that the sample of the colony of microorganisms isdeposited on the depositing spot, in particular covering at mostapproximately half of said one of the depositing spots of the targetplate. Thereafter the target plate with said sample is automaticallytransferred into an apparatus for performing MALDI for identification ofsaid sample of said selected colony of microorganisms.

More detailed this method comprises the following automated steps:

-   -   providing a first picking tool and providing a positioning        device with a picking tool holder for holding a picking tool,        said positioning device being arranged for positioning a picking        tool in a starting position above the obtained location of the        selected colony of microorganisms on the culture dish and for        automatically lowering and raising a picking tool towards and        away from the culture dish and for positioning a picking tool in        a transfer position, respectively;    -   positioning said first picking tool in said picking tool holder        of the positioning device;    -   by said positioning device positioning the first picking tool in        the starting position above the obtained location of the        selected colony of microorganisms on the culture dish,        automatically lowering the first picking tool towards the        culture dish into contact with the microorganism to pick up a        sample of said microorganism, automatically raising the first        picking tool with said sample of the microorganism away from the        culture dish to the transfer position;    -   providing a target plate holder for holding a target plate, said        target plate having a plurality of depositing spots;    -   positioning a target plate in the target plate holder;    -   providing a transferring device for automatically transferring        the picking tool from the transfer position of the positioning        device to a position above one of the depositing spots of the        target plate, and for lowering the picking tool such that the        sample of the colony of microorganisms present on the picking        tool comes into contact with the target plate, moving the        picking tool in a plane parallel to the plane of the target        plate such that the sample of the colony of microorganisms is        deposited on the depositing spot, in particular covering at most        approximately half of said one of the depositing spots of the        target plate; and raising said picking tool from the target        plate. A drop of a MALDI matrix solution is then automatically        laid over on the amount of the sample deposited on the target        plate.

In case the same colony of microorganisms is to be subjected to anotheranalysis or test, such as but not limited to an AntibioticSusceptibility Test (AST) a second sample of said selected colony ofmicroorganisms can be obtained in a manner similar to obtaining theinitial sample. Since the location of the colony of microorganisms onthe culture dish has already been selected, and is therefore “known” tothe central control computer, it is possible to easily and reproduciblyobtain a second sample from the same colony. At least some of the secondsample of said selected colony of microorganisms is then transferred anddeposited on a test culture dish, which can for example be automaticallypositioned at another position below the transfer track 18. Thereaftersaid test culture dish is automatically transferred to an apparatus forperforming a susceptibility test or another additional analysis.

Since for performing characterization and identification ofmicroorganisms normally a plurality of colonies is grown on a culturedish, and in addition a plurality of different culture dishes is used,the invention provides the possibility to identify each culture dishseparately, e.g. by means of a bar code, and furthermore each colony ofinterest on a single culture dish is selected and given anidentification mark. Hereto before the automated step of locating andselecting a colony of microorganisms on a culture dish, the methodaccording to the invention comprises a step of providing a culture dishcomprising a number of colonies of microorganisms, obtaining an initialimage of said culture dish including all the colonies of microorganisms,displaying said initial image of said culture dish including all thecolonies of microorganisms on a display, and selecting at least onecolony of microorganisms in said initial image. In this manner aresearcher or analyst can select colonies of interest based on thorougheducation and knowledge. Since each culture dish is provided with anindividual identification identifying said culture dish, such as a barcode, the initial image of said culture dish including all colonies isstored, and information regarding said at least one selected colony ofmicroorganisms is stored (preferably with links given in the(electronic) initial image), storing all said information andidentifications of the culture dish in a memory of a central controlcomputer allows a very correct further processing. In this manner theonly manual operation is the act of selecting the interesting colonies,whereas all relevant data are processed in an automated way. Optionallythe researcher or analyst can manually enter processing instructionsregarding the processing to which a selected colony of microorganisms ofsaid culture dish is to be subjected, said processing instructions beingalso stored in said memory of said central control computer for lateruse. After this manual act all the further steps are performedcompletely automated in a reliable and efficient manner. For thisautomated further processing the culture dish is automaticallypositioned on a stage for a culture dish of a picking tool devicecomprising an imaging device. An image of said culture dish positionedin said picking tool device is obtained, and together with theidentification of said culture dish it is possible to compare this imageobtained by the imaging device of said picking tool device with thestored initial image of said culture dish and thus derive informationregarding the location of the selected colony of microorganisms andoptionally regarding the processing instructions regarding the processesto be performed on said selected colony of microorganisms. By comparingthe image of the culture dish when it is placed in the picking tooldevice with the initial image, the location of the selected colonies canbe obtained automatically, for example by computerized image comparison.Furthermore each target plate is provided with an identifying mark andoptionally each depositing spot of said target plate has an individualidentification mark or location identifier, so that after storing theidentifying mark of said target plate and depositing spots together withthe properties of the suspension with a link to the identity of theculture dish from which the selected colony of microorganisms wasobtained in the memory of said central control computer a correctlinking of the obtained MALDI results to the specific colony ofmicroorganism under test is possible in a correct and automated way.

Now an embodiment of a method of the invention will be described inwhich a suspension is made from a sample of a colony of microorganismspicked from a culture dish together with an embodiment of an apparatusfor performing such a method.

FIG. 1 schematically shows an embodiment of an apparatus 1 for theautomatic preparation of a suspension of a sample of a microorganismaccording to the invention. Said apparatus 1 comprises a stage 2 for aculture dish 3 comprising a microorganism 4 on a nutritional layer 5,such as a layer of agar gel.

The apparatus 1 further comprises a first picking tool 6 and a furtherpicking tool 7. A positioning device 8 comprises a picking tool holder 9for, in the shown embodiment releasably holding a picking tool, in theembodiment shown in FIG. 1 the picking tool holder 9 holds the firstpicking tool 6. The positioning device 8 is arranged for positioning thefirst picking tool 6 in a starting position (shown in solid lines inFIG. 1) above the culture dish 3 and is arranged for automaticallylowering and raising the first picking tool 6 towards and away from theculture dish 3, such that the first picking tool 6 can be positioned ina position (indicated with broken lines 6′) in which it contacts themicroorganism 4 and picks up a sample of said microorganism. After thefirst picking tool 6 has picked up a sample the positioning device 8raises and positions the first picking tool 6 in a transfer position,which in the embodiment shown in FIG. 1 is identical to the startingposition. In other embodiments the starting and transfer positions maybe different from each other.

The apparatus 1 according to the invention further comprises asuspension tube holder 10 for holding a suspension tube 11 which cancontain a suspension medium which is dispensed from an automaticsuspension medium dispenser 12, which in the shown embodiment has adispensing nozzle 13 for automatically dispensing a suspension medium 14in the suspension tube 11 held in the suspension tube holder 10. In thepresent embodiment the suspension tube holder 10 is a rotatablesuspension tube holder for rotating the suspension tube 11 around avertical axis A.

A transferring device 15 is incorporated in the inventive apparatus 1for automatically transferring a picking tool from the transfer positionof the positioning device 8 to a position above the suspension tube 11held in the suspension tube holder 10. In the embodiment shown thetransferring device 15 comprises a transfer holder 16 with a graspingmeans 17 for releasably holding a picking tool. The transferring device15 may in a manner known per se be mounted on a transfer track 18, suchas a rail, for linear movement thereon as indicated by the arrows. Inthis manner the transferring device 15 may be moved to the positioningdevice 8, such that the grasping means 17 can take over the picking toolfrom the positioning device 8, the picking tool holder 9 thereofreleasing the picking tool after the grasping means 17 has grasped thepicking tool. In the embodiment shown in FIG. 1 the second or furtherpicking tool 7 having previously picked up a sample 19 of themicroorganism 4 is positioned above the suspension tube 11 by thetransferring device 15 in a starting position indicated by solid lines.The transferring device 15 is arranged for lowering the second pickingtool 7 into the suspension medium 14 contained in the suspension tube11, in which position the second picking tool 7′ with the sample 19 issubmerged in the suspension medium 14 as indicated by broken lines inFIG. 1. In this position the transferring device 15 is activated foroscillating the second picking tool 7 in a linear vertical movement fora period of time which is sufficient for the sample 19 to be releasedfrom the second picking tool 7. Thereafter the transferring device 15positions the second picking tool 7 in a waiting position above thesuspension tube 11, which waiting position is in the embodiment shown inFIG. 1 identical to the starting position. In other embodiments thewaiting position and the starting position may be different from eachother.

The inventive apparatus 1 further is provided with a turbidity meter 20for performing measurements of the turbidity of the suspension medium 14contained in the suspension tube 11 held in the suspension tube holder10. As generally known in the art a turbidity meter can providemeasurement values which are a measure of the concentration of material,in the present case the concentration of a microorganism suspended inthe suspension medium. In the embodiment shown in FIG. 1 the turbiditymeter 20 comprises a laser 21 which transmits laser light towards andthrough the suspension medium and a sensor 22 which detects the amountof laser light transmitted through the suspension medium. In additionthere is a further sensor (not indicated in the drawing) which is e.g.arranged perpendicular to the path of the laser light to detect theamount of laser light which has been scattered by the suspension.

The operation of the inventive device is controlled by a controller 23,for example comprising a microprocessor, which is communicativelyconnected (as indicated by the signal lines) to the positioning device8, the transferring device 15, the automatic suspension medium dispenser12, and the turbidity meter 20 for automatically controlling themovement of the positioning device 8, the movement of the transferringdevice 15, the operation of the automatic suspension medium dispenser 12and the operation of the turbidity meter 20, respectively. In additionthe controller 23 might be directly communicatively connected to otherparts of the apparatus such as for example the picking tool holder 9,the transfer holder 16, the laser 21 and the sensor 22.

In the embodiment shown in FIG. 1 the controller 23 is arranged forcontrolling the turbidity meter 20 such that the turbidity measurementof the suspension medium 14 is started before the second picking tool 7is submerged in the suspension medium 14. In addition the controller 23controls the rotatable suspension tube holder 10 for starting therotation of the suspension tube 11 held in the holder 10 before thesecond picking tool 7 is submerged in the suspension medium 14, and formaintaining the rotation of the suspension tube 11 during themeasurement of the turbidity of the suspension medium 14. In the shownembodiment the controller 23 controls the turbidity meter 20 such thatthe measurement of the turbidity is performed during the total period oftime during which the second picking tool 7 is oscillated. In thismanner the turbidity meter 20 provides an on-line measurement value tothe controller 23 which value is indicative of the measured turbidity,and thus the concentration of the microorganism, during the period oftime during which the second picking tool is oscillated.

The controller 23 comprises a memory in which a first and a secondthreshold value are stored, in which said first threshold value is equalto or greater than the second threshold value. If the turbiditymeasurement value provided by the turbidity meter is equal to or betweenthe first and second threshold value, the concentration/amount ofmicroorganism in the suspension medium is sufficient to allow thesuspension tube with the suspension to be further processed. In thatcase the controller 23 provides a signal that the suspension tube can beprocessed further. In addition in this situation the second picking tool7 can be discarded e.g. by transferring the transferring device to aposition C in which the grasping means 17 are activated to release thesecond picking tool 7.

In case the final measurement value of the turbidity meter is above thefirst threshold value previously stored in a memory of the controller 23then the concentration of the microorganism is too high to allow thesuspension tube to be processed further. In that situation thecontroller 23 controls the automatic suspension medium dispenser 12 tosupply an additional amount of suspension medium into the suspensiontube 11. This additional amount of suspension medium is based on theinitial amount of suspension medium, the final measurement value and thevalue of the first and/or second threshold value such that the additionof the additional amount of suspension medium to the suspension mediumalready present in the suspension tube 11 will lead to a concentrationof microorganism in the suspension medium which satisfies therequirement for further processing, as can be confirmed by an additionalor further measurement of the turbidity by the turbidity meter 20.

In case the final measurement value of the turbidity meter 20 is belowthe second threshold value, meaning that the concentration ofmicroorganism in the suspension medium is too low, the controller 23controls the apparatus 1 such that an additional sample of microorganismis picked up by the first picking tool 6 (alternatively the second oranother picking tool can be used for picking up an additional sample).Thus the controller 23 in this case controls the positioning of thetransfer device 15 such that the second picking tool 7 is discarded asdescribed above. Then (or simultaneously) the first picking tool 6 inthe picking tool holder 9 of the positioning device 8 is lowered fromthe starting position above the culture dish 3 towards the culture dishand into contact with the microorganism 4 to pick up an additionalsample of said microorganism. Thereafter the first picking tool 6 isautomatically raised with the additional sample of the microorganismaway from the culture dish to the transfer position. Then saidtransferring device automatically transfers the first picking tool withthe additional sample of the microorganism from the transfer position ofthe positioning device 8 to a position above the suspension tube 11. Thefirst picking tool 6 with the additional sample of the microorganism islowered into the suspension medium 14 and is oscillated by thetransferring device 15 in a linear vertical movement for a period oftime for releasing the additional sample of said microorganism in thesuspension medium. Again the turbidity is measured during theoscillation, and the measured value is compared with the first andsecond threshold value stored in the memory of the controller 23. Inthis case the controller 23 can be arranged for controlling the movementof the transferring device 15 such that the first picking tool 6 israised to the waiting position if during oscillation the on-linemeasurement value of the turbidity performed by the turbidity meter 20is equal to or lower than the first threshold value and equal to orgreater than the second threshold value.

Suspension tubes which are particularly useful in the inventiveapparatus have a substantially circular cross-section with a diameter ofabout 6 to about 12 mm, preferably about 10 mm. In these relativelysmall suspension tubes the controller 23 can control the automaticsuspension medium dispenser 12 such that the supplied initial amount ofsuspension medium is about 0.5-2 ml, preferably about 1 ml.

The oscillation of the transferring device 15 is controlled by thecontroller 23 such that the picking tool oscillates at a frequencybetween about 5 Hz to about 120 Hz, preferably about 30 Hz to about90Hz, most preferably about 50 Hz, with an amplitude of about 0.5 mm toabout 4 mm, preferably about 2 mm to about 3 mm. The controller isfurthermore arranged for controlling the oscillation of the transferringdevice 15 such that the period of time during which the picking tooloscillates is about 3 seconds to about 10 seconds, preferably about 6seconds.

The inventive apparatus 1 in addition comprises a conveyor 24 of whichthe end position can form the stage 2 for the culture dish or as shownin FIG. 1 a conveyor 24 and a stage 2 which are mutually positioned suchthat a culture disk can be transported onto the stage and removed fromthe stage by appropriate operation of the conveyor 24. The conveyor 24is controlled by the controller 23 for automatically positioning andremoving a culture dish comprising said microorganism on and from thestage, respectively. Please note that in other, not shown, embodimentsdifferent means for automatically positioning and removing a culturedish on and from the stage, respectively, can be used. In particular thecontroller 23 is arranged for allowing a culture dish to beautomatically removed from the stage by the automatic culture dishpositioning and removing device only after the signal that thesuspension tube with the suspension can be removed from the suspensiontube holder for further processing has been provided. This ensures thatit is always possible to pick up an additional sample, if necessary.

As shown in FIG. 1 the inventive apparatus 1 further comprises anautomatic suspension tube positioning and removing device 25 forautomatically positioning and removing a suspension tube in and from thesuspension tube holder, respectively. In the shown embodiment automaticsuspension tube positioning and removing device 25 comprises graspingmeans 26 for releasably grasping a suspension tube 11′. Again, thecontroller 23 is arranged for being communicatively connected to theautomatic suspension tube positioning and removing device 25, 26 forcontrolling the operation of the automatic suspension tube positioningand removing device, and for automatically positioning a suspension tubein the suspension tube holder. The controller 23 in particular isarranged for automatically removing a suspension tube holder from thesuspension tube holder by the automatic suspension tube positioning andremoving device 25, 26 only after the signal that the suspension tubewith the suspension can be removed from the suspension tube holder forfurther processing has been provided. In the shown embodiment theautomatic suspension tube positioning and removing device 25 is movablealong the rail 18 independent of the movement of the transferring device15. In the position indicated by C empty suspension tubes can be fetchedand suspension tubes with a suspension medium containing a sufficientconcentration of microorganism can be handed over to equipment forfurther processing, such as an incubator. Please note that the positionC can for example be formed by a multitrack system which can lead thesuspension tube positioning and removing device 25 and the transferringdevice 15 to different location at which different components arepresent or processes can be performed.

The sample suspension thus prepared is used for performingcharacterization or identification of the microorganisms using MALDI andoptionally used for other analysis, such as AST. For identifyingmicroorganisms using MALDI a drop of said sample suspension is obtained;and this drop is transferred onto said target plate. A drop can beobtained by using another picking tool which is held by the graspingmeans 17 and which is automatically lowered in the suspension. When thispicking tool is raised out of the suspension a drop of suspension willstick on the tip of this picking tool, which can be transferred alongthe track to position B, where the picking tool with the drop is lowereduntil the drop contacts the depositing spot on the target plate, and atleast a part of the drop will remain on the depositing spot after thepicking tool has been raised away from the target plate. Alternatively apipetting tool, to be described below, can be used to pick up an amountof suspension from the suspension tube, transfer this amount to positionB and deposit a drop of suspension on the target plate. After a drop ofsuspension has been deposited on the target plate, and in particularwhen this drop has been allowed to dry, a drop of a MALDI matrixsolution is automatically overlaid on the amount or portion of thesample deposited on the target plate. For performing other tests oranother analysis a second drop of said sample suspension can be obtainedin a similar way, and such a drop may be automatically transferred toand deposited on e.g. a test culture dish which is further transferringin an automated way for performing a susceptibility test or anotheradditional analysis.

Each suspension tube comprises an unique identifying mark, which isstored together with the properties of the suspension with a link to theidentity of the culture dish from which the selected colony ofmicroorganisms was obtained in the memory of said central controlcomputer for the purpose of amongst other things correctly and in a fastmanner link the obtained results of analysis with the culture dish andcolony pertaining to said results.

Although not shown in FIG. 1 an amount of suspension can be taken fromthe suspension in the suspension tube by means of a pipetting tool whichcan be automatically held and positioned by the grasping means(functioning as pipetting tool holder) 17 and transferring orpositioning device 15 in the same manner as a picking tool. Thepositioning device 15 is arranged for positioning said pipetting tool ina starting position above the suspension tube and for automaticallylowering and raising the pipetting tool into and out of the suspensionand for positioning the pipetting tool in a transfer position,respectively. When the pipetting tool is lowered into the suspension insaid suspension tube the pipetting tool is operated in a manner knownper se (e.g. using sub pressure) to pick up an amount of suspension.Thereafter the pipetting tool with said amount of suspension is raisedto the transfer position. For holding the amount the pipetting toolcomprises a pressurizable chamber closed by a controlled valve. Thepipetting tool is automatically transferred by the transferring device15 to the position B above one of the depositing spots of the targetplate. In this position the pipetting tool is lowered to a predefineddistance above the target plate, after which the chamber is pressurizedto a pressure in a range of about 0.5 bar to 1.1 bar. The valve is thenopened for such a time that a drop of suspension with a volume in arange of about 0.5 to 3.0 μl is deposited on the depositing spot, inparticular covering at most approximately half of said one of thedepositing spots of the target plate. After the drop has been depositedthe pipetting tool is raised from the target plate and can betransferred to position C where it can be discarded or cleaned forreuse.

1-50. (canceled)
 51. An apparatus for automatic preparation of asuspension of a sample of microorganisms, said apparatus comprising: astage for a culture dish comprising said microorganism; a first and afurther picking tool and a positioning device with a picking tool holderfor holding a picking tool, said positioning device being arranged forpositioning a picking tool in a starting position above the culture dishand for automatically lowering and raising a picking tool towards andaway from the culture dish and for positioning a picking tool in atransfer position, respectively; a suspension tube holder for holding asuspension tube; an automatic suspension medium dispenser forautomatically dispensing a suspension medium in a suspension tube heldin the suspension tube holder; a transferring device for automaticallytransferring a picking tool from the transfer position of thepositioning device to a position above a suspension tube held in thesuspension tube holder, and for lowering and raising a picking tool intoand away from a suspension medium contained in a suspension tube, andfor positioning a picking tool in a waiting position above a suspensiontube held in the suspension tube holder, respectively, said transferringdevice further being arranged for oscillating a picking tool in a linearvertical movement for a period of time; a turbidity meter for performingmeasurements of the turbidity of a suspension medium contained in asuspension tube held in the suspension tube holder and for providing afinal measurement value indicative of the measured turbidity; acontroller communicatively connected to the positioning device, thetransferring device, the automatic suspension medium dispenser and theturbidity meter for automatically controlling the movement of thepositioning device, the movement of the transferring device, theoperation of the automatic suspension medium dispenser and the operationof the turbidity meter, respectively; said controller being arrangedfor: a) determining whether the final measurement value is above a firstthreshold value previously stored in a memory of the controller, if yessaid controller being arranged for performing step b); or whether thefinal measurement value is identical to or below the first thresholdvalue and identical to or above a second threshold value previouslystored in the memory of the controller, said first threshold value beingequal to or greater than the second threshold value, if yes saidcontroller being arranged for performing step c); or whether the finalmeasurement value is below the second threshold value, if yes saidcontroller being arranged for performing step d); b) controlling theautomatic suspension medium dispenser to supply an additional amount ofsuspension medium into the suspension tube; c) providing a signal thatthe suspension tube with the suspension can be removed from thesuspension tube holder for further processing; or d) positioning saidfurther picking tool in said picking tool holder of the positioningdevice; by said positioning device positioning the further picking toolin the starting position above the culture dish, automatically loweringthe further picking tool towards the culture dish into contact with themicroorganism to pick up an additional sample of said microorganism,automatically raising the further picking tool with said sample of themicroorganism away from the culture dish to the transfer position; bysaid transferring device automatically transferring said further pickingtool with the additional sample of the microorganism from the transferposition of the positioning device to a position above the suspensiontube held in the suspension tube holder, lowering said further pickingtool with the additional sample of the microorganism into the suspensionmedium contained in the suspension tube; by the transferring deviceoscillating the further picking tool in a linear vertical movement for aperiod of time while the further picking tool with the additional sampleof said microorganism is submerged in the suspension medium; and aftersaid period of time has elapsed raising the further picking tool awayfrom the suspension medium contained in the suspension tube to thewaiting position; at least after the period of time during which thefurther picking tool is oscillated has elapsed measuring the turbidityof the suspension medium contained in the suspension tube held in thesuspension tube holder by the turbidity meter and providing anadditional final measurement value indicative of the measured turbidity;and performing step a).
 52. An apparatus for the automatic preparationof a suspension of a sample of microorganisms according to claim 51,wherein the controller is arranged such that the step of measuring theturbidity of the suspension medium contained in the suspension tube heldin the suspension tube holder by the turbidity meter is additionallyperformed during the period of time during which the picking tool isoscillated, wherein the turbidity meter is arranged for providing anon-line measurement value indicative of the measured turbidity duringthe period of time during which the picking tool is oscillated to thecontroller.
 53. An apparatus for the automatic preparation of asuspension of a sample of microorganisms according to claim 52, whereinthe controller is arranged for controlling the movement of thetransferring device such that a picking tool is raised to the waitingposition if during oscillation the on-line measurement value of theturbidity is equal to or lower than the first threshold value and equalto or greater than the second threshold value, and for providing asignal that the suspension tube with the suspension can be removed fromthe suspension tube holder for further processing.
 54. An apparatus forthe automatic preparation of a suspension of a sample of microorganismsaccording to claim 52, wherein the controller is arranged forcontrolling the turbidity meter such that measuring the turbidity of thesuspension medium contained in the suspension tube held in thesuspension tube holder by the turbidity meter is started before thepicking tool is submerged in the suspension medium contained in thesuspension tube. 55-60. (canceled)